A New Quantification Method of Bacterial Adherence on Implant Surfaces in an Implant Associated Infection Rat Model

Abstract

Objective: Implant infections in elective orthopedic surgery are still a clinically relevant problem with devastating consequences for the patient. Thereof results a great need for new strategies to prevent implant infections. Functionalization of implant surfaces to reduce the microbial adherence show great potential in vitro, but have to be tested in suitable models in vivo. Proper evaluation methods of the bacterial load on the implant surface are important for their evaluation. Up to now, the simultaneous assessment of the quantity and morphology of the bacterial infection in vivo was not performed. Methods: Cubic Ti90/Al6/V4-rods were inserted in the tibia of Lewis rats and infected with Staphylococcus aureus strain 36/07 in different concentrations. After 21 days, explanted implants were stained for living and dead cells. The bacterial surface colonization was analyzed by confocal microscopy and assessed semi quantitatively via two different approaches (spot method and volume method) using the software Imaris ×64. Furthermore the bacterial morphology was evaluated. The results were compared to radiographic and histological changes. Results: The new semi quantitative CLSM evaluation to assess the bacterial biomass on implant surfaces was successfully implemented. Both methods gave equivalent results. The results of the morphologic assessment of the bacterial colonization were similar to those of the quantification. A tendency towards increasing bacterial biomass and biofilm formation on the implant surface was observed with decreasing infection concentrations. In contrast, histologic and radiographic assessment as well as the relative tibial bone weight revealed more severe changes for higher inoculation concentrations. Conclusion: In combination with the morphological assessment of the bacterial appearance this CLSM based evaluation is a suitable tool to assess the bacterial load on the implant surface. Combined with radiographical and histological evaluation of bone alterations, this model is appropriate for the evaluation of new implant surfaces.